Cyclic GMP Phosphodiesterase in Signaling

描述(由申请人提供)： 细胞内环核苷酸水平的调节是细胞信号传递的主要模式，尤其是通过G蛋白偶联受体(GPCRs)传递信号。最近，我们已经证明了包括Frizzleds在内的5个7-跨膜节段性受体家族中的两个成员是关于几个下游信号通路的GPCRs。两个观察结果为这项建议的具体目标提供了基础：抑制小鼠F9畸胎瘤干细胞中的异源三聚体G蛋白亚基G(α)T2/o，以及环状GMP PDE的抑制剂，如IBMX、扎匹司特和潘生丁，阻断了GPCR大鼠Fz2在钙瞬变和环状GMP水平上的信号传递能力。我们已经创造了一个嵌合受体，由广为人知的GPCR(β)2-肾上腺素能受体的跨膜、配体结合结构域和外部片段组成，三个细胞内环(iloop1-3)和Rfz2的C末端、细胞质尾巴已经拼接到该受体上。这种新型嵌合体与β-肾上腺素能激动剂和拮抗剂结合，发出钙动员和降低细胞内环鸟苷浓度的信号，但不能与G(α)S和腺苷环化酶结合，就像野生型(β)2-肾上腺素能受体一样。我们已经验证了构建物的功能，并提出了两个特定的目的来以生物化学的方式阐明G(α)T2/o和环GMP PDE在信号转导中的新作用：通过杆状病毒诱导的这些成分的表达和BRET在F9细胞中的表达，生物化学检测FZ2和在Sf9细胞中表达的异源三聚体G蛋白之间的直接相互作用；通过体外表达、纯化和重组受体/G-蛋白/PDE三联体以及F9细胞中的反义抑制/挽救和BRET分析来确定负责这一信号转导的PDE(S)的分子身份。我们将在这些细胞中使用一种新的药物诱导分泌系统来验证(使用天然配体和受体)利用嵌合体进行的观察。这些研究与信号转导以及阐明人类疾病的基础高度相关，在这些疾病中，信号转导途径的改变转化为异常生物学。

DESCRIPTION (provided by applicant): Regulation of intracellular levels of cyclic nucleotides is a major paradigm in cell signaling and, in particular, signaling by G-protein-coupled receptors (GPCRs). Recently, we have shown that two members of the family 5 of 7-transmembrane segmented receptors that includes Frizzleds are GPCRs with respect to several downstream signaling pathways. Two observations provide the basis for the specific aims in this proposal: suppression of the heterotrimeric G-protein subunits G(alpha)t2/o in mouse F9 teratocarcinoma stem cells and treatment with inhibitors of cyclic GMP PDE, such as IBMX, zaprinast, and dipyridamole, block the ability of the GPCR rat Fz2 to signal at the level of calcium transients and cyclic GMP. We have created a chimeric receptor composed of the transmembrane, ligand-binding domain and the exofacial segments of the well-known GPCR (beta)2-adrenergic receptor to which the three intracellular loops (iloops 1-3) and the C-terminal, cytoplasmic tail of Rfz2 have been spliced. This novel chimera binds beta-adrenergic agonists and antagonists, signaling to calcium mobilization and reduction in intracellular concentrations of cyclic GMP, but not to G(alpha)s and adenylylcyclase, like the wild-type (beta)2-adrenergic receptor. We have validated the functional capability of the construct and propose two specific aims to elucidate in a biochemical manner a new role for G(alpha)t2/o and cyclic GMP PDE in signaling: to test for the direct interaction between Fz2 and heterotrimeric G-proteins expressed in Sf9 cells biochemicaly by baculovirus-induced expression of these components and by BRET in F9 cells; and, to establish the molecular identity of the PDE(s) responsible for this signaling by expression, purification, and reconstitution of the triad of receptor/G-protein/PDE in vitro and complementary studies in F9 cells using antisense suppression/rescue as well as BRET analysis of protein-protein interactions. We shall employ a novel drug-induced secretion system in these cells to validate (with native ligand and receptor) the observations exploited by use of the chimera. These studies are highly relevant to signaling as well as to elucidation of basis for human diseases in which alterations in signaling pathways translate into aberrant biology.